We investigated the participation of the STAT pathway in A. gambiae anti-plasmodial responses. STAT silencing reduces the number of early Plasmodium oocysts that form in the midgut, but paradoxically enhances the overall infection by increasing oocyst survival. Silencing of SOCS, a STAT suppressor, has the opposite effect and reduces Plasmodium infection through the activation of Nitric Oxide Synthase (NOS) expression. All other immune pathways described so far in An. gambiae affect Plasmodium soon after midgut invasion is completed, at the ookinete-to-oocysts transition. The current dogma has been that once oocysts mature and modify their surface, they are hidden and unaffected by the mosquitos immune system. Our findings established that this is not the case, and defined a novel late phase in the mosquito antiplasmodial responses. The AgSTAT-A pathway mediates this late response that decreases oocyst survival. Recent experiments indicate that adult mosquito females challenged with Plasmodium are able to learn and respond more efficiently to subsequent challenges. This effect is long lasting and both the priming and the recall require ookinete invasion of midgut epithelial cells in the presence of bacteria from the midgut flora. Preliminary data showed that the priming response involves hemocyte differentiation that results in a long-lasting increase in the number of circulating granulocytes, the major cells involved in bacterial phagocytosis. We are currently comparing the transcriptional profile of naive and challenged hemocytes using Solexa high throughput sequencing. Circulating hemocytes respond to ookinete invasion by attaching to the basal surface of the midgut. Preliminary data indicate that a greater number of hemocytes adhere to the midgut when primed females are re-infected than in naive controls. We identified a heme peroxidase that is secreted into the midgut lumen and prevents the activation of antibacterial and antiplasmodial immune responses of midgut epithelial cells, allowing proliferation of the bacterial flora and Plasmodium development. IMPer silencing dramatically reduces P. berghei and P. falciparum infection and induces high levels of nitric oxide synthase (NOS) expression. Furthermore, double silencing of IMPer and NOS rescues parasite survival to control levels. This physiological induction of IMPer following a blood meal provides a permissive environment for Plasmodium development, as it prevents activation of both antibacterial and NOS-mediated anti-malarial responses. We recently found that the Dual Oxidase (Duox) enzymes provides hydrogen peroxide to IMPer, and together they catalyze a cross-linking reaction that results in a dynamic barrier that modulates the permeability of the mucosal layer to immune elicitors. We identified a serine protease (SP30) that is requires for Plasmodium parasites to efficiently invade midgut epithelial cells. SP30 silencing also abolished the induction of the serine protease SRPN6 mRNA in response to Plasmodium midgut infection, suggesting that both genes could be part of the same signaling cascade, with SP30 acting upstream of SRPN6. The mechanism by which SP30 silencing affects ookinete midgut invasion is under investigation. SP30 appears to be part of a general signaling cascade triggered by Plasmodium invasion, as SP30 expression is also highly induced when mosquito salivary glands are invaded by Plasmodium sporozoites. Furthermore, SP30 silencing significantly decreases the number of sporozoites that invade the salivary gland. The E. coli expression system was used to produce recombinant SP30, which was purified and injected into rabbits to generate polyclonal antiserum. These antibodies recognize a single band in midguts of sugar-fed females and their specificity will be further confirmed by silencing SP30 expression. The subcellular localization of SP30 will be determined by immunofluorescence staining of midguts from mosquitoes fed on either a healthy or a Plasmodium-infected mouse. We are collaborating with Dr. Jose Ribeiros group to re-fold SP30 and investigate its substrate specificity, enzyme kinetics and interaction with specific inhibitors. A Drosophila genetic screen identified several genes that affect P. gallinaceum infection in the fly. We compared how silencing a set of genes identified in this screen affects Plasmodium infection in different mosquito-parasite combinations. We conclude that there is a broad range of compatibility between different Plasmodium strains and particular mosquito strains. Compatibility is defined as the extent to which the immune system of the mosquito is actively limiting Plasmodium infection. A strain can be highly compatible either because of the genetic background of the vector (as mosquitoes can be selected to be either highly susceptible or refractory to infection) or due to the parasites ability to evade the mosquito immune system. For example, the P. yoelii-An. stephensi and P.falciparum-An. gambiae strains used in this study are highly compatible combinations, and silencing several genes involved in oxidative response or immunity has no significant effect on infection. In contrast, silencing the same genes has a strong effect in less compatible vector-parasite combinations such as P. yoelii-An. gambiae or P. berghei-An. gambiae. The oxidation resistance 1 (OXR1) gene is highly conserved between species and is known to protect yeast, rodent and human cells from oxidative stress through an unknown mechanism. Gene silencing experiments in An. gambiae adult females revealed that OXR1 regulates expression of catalase and glutathione peroxidase, two critical enzymes that detoxify hydrogen peroxide (H2O2);and provided the first experimental evidence that OXR1 expression is regulated by the JNK pathway. OXR1 is essential for efficient reactive oxygen species (ROS) detoxification, as silencing this gene significantly decreases the survival of adult females to an oral challenge with H2O2. OXR1 silencing also dramatically decreases the number of P. berghei ookinetes that survive and transform into oocysts, in agreement with previous studies from our group indicating that ROS are key effectors of mosquito immune responses against bacteria and Plasmodium. The unexpected observation that OXR1 silencing does not affect P. falciparum infection suggests that this parasite species may be less susceptible to ROS damage. We identified a QTL in Chr. 13 that confers an African strain of Plasmodium falciparum (GB4) the ability to survive in An. gambiae (L35) females. In contrast, 98-100% of P. falciparum 7G8 parasites (Brazilian strain) are detected by the mosquito immune system and killed. The melanization phenotype in females infected with 7G8 can be completely reversed by silencing genes known to mediate antiplasmodial responses, indicating that these two P. falciparum strains differ in their ability to evade the mosquitos immune system. The QTL encompasses a 171.8 kb region coding for 42 genes. Multiple alternative strategies will be used to identify the gene that confers this phenotype. In collaboration with Dr. Sus Unit, we analyzed genomic tile arrays from the parental and progeny lines and identified 13 candidate genes in the QTL regions that have single motif polymorphisms (SMPs) that correlate with survival of the progeny lines. These polymorphisms will be confirmed by direct sequencing. This will also make it possible to identify differences in the deduced amino acid sequence of these genes. The mRNA expression levels of the candidate genes in the ookinete and gametocyte stages of the parasite will be determined, to rule out genes that are not expressed in these stages of the parasite. Expression levels (mRNA) of all candidate genes in the two parental strains will also be compared.